RECOMBINANT PROTEIN FOR BIOCONTROL OF RALSTONIA SOLANACEARUM . S.S. Kabeil, Elsayed E. Hafez, Ayman S. Daba and A.
El-Saadani
Mubarak City for Scientific Research and Technology, Borg-Elarab, Alexandria, Egypt.
Abstract: In Egypt, potato has an important position among all vegetable crops, where
about 20% of total area devoted for vegetable production is cultivated with
potato. Recently, potato crop is infected with the brown rot disease producing
a major problem which caused by Ralstonia solanacearum. Soil samples
were collected from Gharbia governorate, bacterial isolation was carried out.
Many bacterial isolates were obtained and used for bioassay against the R.
solanacearum. One isolate showed high activity against the pathogenic
bacteria R. solanacearum, subjected to identification using the 16S rRNA
gene and the sequence analysis revealed that the strain is Pantoea
agglomerance. The bacteria was cultivated on PBG medium and the culture
filtrate was fractionated and the fraction were used to control such bacteria.
One of these fractions showed high ability to control the pathogenic bacteria
(named Biocine). The Molecular weight of the purified Biocine was
determined by SDS polyacrylamide gel electrophoresis revealing one band
with a molecular weight of 29kDa. Differential display was used to study the
gene regulating the production of Biocine in the isolated strain using arbitrary
primer. Band in molecular weight 800bp were obtained, cloned using TOPO
TA cloning kit. The fragment was released using the EcoR1 restriction
enzyme and subcloned into prokaryotic expression vector (PtracA). The
recombinant bacteria was induced using the IPTG and the purified protein
was obtained based on the His-tag technology. Bioassay was carried out for
the purified recombinant protein compared with the wiled one, the same
activity was shown. Now we can concluded that we are able to produce a
high amount of biocine on semi-industrial scale and we will try to transfer the
experiment from lab to the field.